Immunosuppression, whether induced by drugs or disease, can lead to alterations in T cell and/or accessory cell function. For example, it has been demonstrated that AIDS patients manifest defective responses to mitogens, autoantigens, alloantigens, and soluble antigens. This altered immunoreactivity is attributable to defects in both responding (T) and stimulating (monocyte and dendritic) cell populations. Decreased CD4 expression on the monocytes obtained from AIDS patients has been demonstrated, yet no decrease in monocyte count has been observed. Immunosuppressive drugs can also alter antigen presenting cell function. As a result of these observations, it has been determined that a method for specifically stimulating T cells which did not rely on the presence of monocytes or dendritic cells would be desirable.
While there are numerous methods of activating T cells, the optimal method appears to require the multivalent interaction of antibodies, or other receptor binding species such as lectins, with the T cell antigen receptor/CD3 complex [hereafter TCR/CD3] on the surface of T cells. [A. Altman et al., Crit. Revs. in Immunol. 10:347-391 (1990)]. CD3 specific monoclonal antibodies can induce highly purified, resting T cells to proliferate, provided, however, that there is present a mechanism for crosslinking the antibody-bound TCR/CD3 complexes. Numerous authors have shown that the crosslinking requirement can be met by binding the antibody to a substrate, for example, Sepharose.RTM. beads [S. Meuer et al., J. Exp. Med. 158:988-999 (1983) and D. A. Hafler et el., J. Immunol. 142:2590-2596 (1989)], polystyrene beads [S. Panzer et al., Scand. J. Immunol. 32:359-371 (1990)], or tissue culture dishes. International Patent Publication WO 90/04633 describes solid-state supported monoclonal antibodies for induction of T cell activation and the growth of T cells. Accessory cells, for example, monocytes, can also fulfill the need for crosslinking by means of Fc receptor mediated binding of the T cell bound anti-CD3 monoclonal antibodies to the monocyte cell surface.
The parameters used to assess cellular activation were reviewed by A. Altman et al., Crit. Revs. in Immunol. 10:347-391 (1990). Cell activation has been measured by changes in nucleic acid synthesis, protein or glycoprotein synthesis, cellular size and morphology, membrane integrity, expression of cellular constituents, cell function, cell growth, cell differentiation and the release of cellular components. These cellular changes have been detected by numerous different methods, many of which are described in the patents and publications cited herein. Historically, the diagnosis of immune deficient conditions has been done using laboratory tests in which various stimuli are applied to T cells in order to determine if they can be activated in vitro. Deficient T cell reactivity has been tested by stimulation with mitogens, alloantigens and soluble antigens [R. Hong in Manual of Clinical Immunology, 2nd Ed., N. R. Rose and H. Friedman, eds. (American Society for Microbiology 1980), Chapter 111, pages 833-849]. However, these methods are not specific for all T cells. Mitogens activate both T and B cells. Alloantigens activate only those selected T cells which have the appropriate receptor type. The response to soluble antigens, for example, tetanus toxoid, can be effected by a patient's immunization history. The anti-CD3 aminodextran conjugates of the claimed invention specifically activate all CD3 positive cells and thus avoid these problems. The CD3 antigen is found on virtually all mature peripheral T lymphocytes. This antigen is a component of the T cell receptor complex and is non-covalently linked to a polymorphic, clonotypic structure termed Ti. Antibodies to the CD3 surface structure serve as probes for constant regions of the T cell receptor which is exclusively expressed on immunocompetent T lymphocytes. Consequently, quantifying immunocompetent T cells using this antibody is rapid and efficient.
In addition to the forementioned copending applications, particles coated with dextran or dextran derivatives have been described by R. J. Mrsny et el., Eur. J. Cell. Biol. 45:200-208 (1987) (ouabain-aminodextran-gold particles); J. W. M. Bulte et al. Magn. Reson. Med. 25:148-157 (1992) (biotinylated dextran-magnetite particles). The use of antibody-dextran type substances as carriers has been described by U. Manabe etal., J. Lab Clin. Mad. 104:445-454 (1984) (antibody-polyaldehyde dextran-methotrexate); A. R. Oseroff et al., Proc. Natl. Acad. Sci. USA 83:8744-8748 (1986) (antibody-aminodextran-chlorin); and S. Rakestraw et al., Proc. Natl. Acad. Sci. USA 87:4217-4221 (1990) (antibody-dextran hydrazide-Sn(IV) chlorin). Other conjugated and crosslinked species have been described by S. S. Wang in "Chemistry of Protein Conjugation and Crosslinking" (CRC Press, Boca Raton, Fla. 1991) and H. Maeda et al., Bioconjugate Chem. 3:351-362 (1992). The standard procedure for the introduction of amine groups into dextran has been to first cleave the sugar rings to form polyaldehyde-dextran. The second step is to react the cleaved rings with a diamine such as ethylenediamine or 1,3-diaminopropane to form a Schiff's base complex. The Schiff's base is then stabilized by reduction with sodium borohydride. The "aminodextran" compounds as described in the above cited art were ill-described, typically lacking either elemental analyses or even average molecular weight determinations. Furthermore, the periodate oxidation method of preparing aminodextrans as described in these publications resulted in a low percentage of amino groups per molecule. The percentage was less than 4-5 percent. Higher degrees of amine substitution were not possible under the usual conditions of the prior art because high diamine concentrations caused extensive aminolysis of the glucosidic linkages between the sugar rings in dextran which resulted in very low molecular weight fragments. As a result, the yields of polymeric aminodextran derivatives were low and decreased drastically as higher and higher degrees of amine substitution were pursued.
An alternative method of producing aminedextrans is by carboxymethylation of sugar residue hydroxyl groups in chloroacetic acid, followed by carbodiimide coupling of a diamine such as ethylenediamine. M. Brunswick et al., J. Immunol 140:3364-3372 (1988) and P. K. A. Mongini et al., J. Immunol. 148:3892-3902 (1992) used this method to produce an aminodextran having about one amine group per sixty-seven glucose residues (1/67). These authors then used the aminodextrans to prepare anti-Ig antibody-aminodextran conjugates for use in inducing B cell activation and proliferation.
The claimed invention teaches the use of anti-CD3 monoclonal antibodies conjugated to aminodextrans as a method of providing for the specific stimulation of T cells. Analysis of T cell function is critical to the diagnosis of immunodeficiency. For example, the CD3-aminodextran conjugates described herein provide a uniquely specific method for activating the T cells used for T cell analysis in AIDS patients. M. Clerci et al., J. Clin. Invest. 84:1892-1899. (1988) found that an in vitro T helper cell (T.sub.H) assay "can detect multiple stages of immune dysregulation early in the course of HIV infection". Tetanus toxoid and alloantigens, which have selectivity problems as stated above, were used as stimuli for T cells. S. C. Muluk et el., Transplantation Proceedings 23:1274-1276 (1991) have shown that T cell monitoring can be useful for determining the efficacy of immunosuppressive agents in transplant patients.
The claimed invention teaches the use of aminodextrans, particularly aminodextrans having a high degree or percentage of amine substitution, as a means of crosslinking antibodies and of using the resulting antibody-aminodextran conjugate to induce activation and proliferation of T cells. Aminodextrans have been used in the copending applications cited herein to coat polystyrene microspheres and magnetic and non-magnetic particles such as ferrite and metallic gold particles. The aminodextran coated particles are then used to covalently link various monoclonal antibodies. Both the degree of amine substitution and the degree of polymerization of the dextran can be varied to determine the optimal form of the resulting coated particle to which an antibody can be conjugated. Non-specific interactions between antibody-aminodextran coated particles and cells are minimized by blocking amine groups with excess crosslinking agent which, in turn, is also blocked.